Protein detection reagents and methods with dyes and dextrins

ABSTRACT

The invention provides reagents, methods and kits for detection of proteins and quantitative determination of protein concentration. The reagents comprise a protein-complexing dye, such as a Coomassie dye and one or more dextrins, for the elimination of interference caused by detergents.

TECHNICAL FIELD

The present invention relates to reagents, methods and kits fordetection of proteins and quantitative determination of proteinconcentration.

BACKGROUND ART

Several methods are available for detecting proteins and determining theconcentration of a protein in solution. These include dye-bindingmethods, which are well known in the art, and involve a non-specificreaction in which a protein-complexing dye binds to the protein. Theformation of a dye-protein complex causes a change in the opticalproperties of the dye, such that there is a colour change proportionalto the amount of protein present in the sample. Protein-complexing dyesused for in vitro protein quantitation include bromocresol green(Gindler, U.S. Pat. No. 3,884,637), HABA and methyl orange, but theseare of limited use as they bind almost exclusively to albumin andgenerally are not very sensitive.

Other methods to determine protein concentration include the Biuretmethod (Mokrasch and McGilvery, J. Biol. Chem. (1956). 221, p. 909), inwhich peptide structures containing at least two peptide linkages arereacted with Cu²⁺ in alkaline solution to form a violet-coloured chelatecomplex.

Lowry et al. (J. Lab. Clin. Med. (1951). 39, 663) used a pre-treatmentof proteins with an alkaline copper solution, similar to the Biuretmethod, followed by addition of Folin-Ciocalteu reagent (which containslithium salts of phosphotungstic and phosphomolybdic acids). The colourproduced was a result of the reduction of the phosphotungstic andphosphomolybdic acids to tungsten and molybdenum blue by the Cu-proteincomplex and by the tryptophan and tyrosine of the protein.

A serious drawback of both the Biuret and Lowry methods is that theycannot tolerate reducing agents that are often present in proteinsamples.

Dye/protein complex formation using Coomassie Brilliant Blue G-250 as aprotein-complexing dye has been described (Bradford U.S. Pat. No.4,023,933). Coomassie Brilliant Blue dyes will bind to a wide variety ofproteins. Moreover, the use of the G-250 dye in the appropriate acidmedium results in a protein assay reagent having a sensitivityapproximately 100 times greater than the Biuret and conventional dyebinding techniques and about 3 to 5 times that of the Lowry method(Bradford U.S. Pat. No. 4,023,933). The use of Coomassie Brilliant BlueG-250 dye in the procedure disclosed in U.S. Pat. No. 4,023,933, the“Bradford Assay”, has many advantages over methods that employ otherdyes, including high sensitivity, which permits the use of small samplesize and utility when reducing agents are present in a sample.

Coomassie Brilliant Blue G-250 exists in two different colour forms, redand blue. The blue form of the dye is present in neutral and alkalinesolution while the red form is present in markedly acid solution (pH0-1). In acidic solution, Coomassie Brilliant Blue G-250 is present inequilibrium between the red and blue forms; such solutions are brownishin appearance. It is believed that as protein binds to the dye, the dyeis brought into a different microenvironment and is then protected fromthe acid medium that gives the red colour to the dye. The strength ofthe acid medium is important for protein assay sensitivity usingCoomassie dyes, because an increase in the strength of the acid mediumcauses a significant loss in sensitivity of the assay. The protein-dyecomplex tends to aggregate, which affects the stability of the colourproduct. The presence of a solubilising agent, such as ethanol, tends tokeep the protein-dye complex from aggregating for a reasonable period oftime; however, too much ethanol results in a marked shift to the blueform of the dye, i.e., change of the environment to one which is lesspolar. It has been postulated that the mechanism of the assay is thebinding of a carbanion form of the dye to a less polar environment ofthe protein. This perhaps also explains the negative effect of largequantities of detergent and of acetone on the assay, since thesecompounds are generally non-polar in nature and would tend to change theenvironment of the dye.

The principal drawbacks of the Bradford assay are the effective lack ofcolour stability for extended periods, largely due to precipitation ofthe protein-dye complex; the failure to show substantially the samereactivity to different proteins; the failure to follow Beer's law; and,most importantly, the adverse affect on the assay of detergents presentin a sample (Bradford, M., Anal. Biochem., 72 248-254, 1976 and U.S.Pat. No. 4,023,933).

Dye/protein complex formation is also used for staining proteins ingels, such as those used in electrophoresis. For example, the dyeCoomassie Brilliant Blue G-250 in perchloric acid solution has been soused (Reisner, A. H. et al. (1975) Anal. Biochem. 64, 509-516).

Currently, several commercial Coomassie-based formulations are availableto stain proteins in gels after electrophoretic separation. For manyelectrophoretic applications, detergents such as SDS are used tofacilitate separation of proteins. Because detergents adversely affectthe colour change on binding of Coomassie dyes to protein, the detergentmust be removed by several wash procedures, resulting in extended andconvoluted staining procedures.

Thus a major disadvantage of dye-based protein detection andquantitation, in particular using Lowry assay reagents or Coomassiedyes, is the interference from detergents, surfactants and otheramphipathic molecules.

Accordingly, there is a desire for reagents and methods for detectionand quantitative determination of protein which have improved toleranceto the presence of detergents in the samples and which have improvedprotein-dye colour stability.

DISCLOSURE OF THE INVENTION

The invention provides a reagent for detection of protein comprising, orconsisting of:

-   -   (a) a protein-complexing dye, and,    -   (b) one or more dextrins.

The protein-complexing dye is a dye which typically undergoes a changein optical properties on formation of a protein-dye complex, this can bea change in absorption spectra as occurs with Coomassie™ brilliant bluedyes, bromocresol green, HABA, methyl orange, Biuret reagent, Biuretreagent with Folin-Ciocalteu reagent (Lowry reagents); or a change inemission spectra, as occurs for dyes which form a fluorescentprotein/dye complex, e.g. Coomassie Orange™, fluorescein, Alexofluor,phycoerythrin, Texas Red™.

It is preferred that the protein-complexing dye does not comprise aprotein, preferably the protein-complexing dye does not comprise anantibody or peptide.

The protein-complexing dye is preferably a Coomassie dye, such as aCoomassie brilliant blue dye, e.g. Coomassie brilliant blue dye G-250 orCoomassie brilliant blue dye R-250. For some protein complexing dyes,and in particular Coomassie dyes, a low pH is required to achieve thenecessary change in optical properties on protein/dye complex formation.

Accordingly, the invention further provides a reagent for detection ofprotein comprising:

-   -   (a) a protein-complexing dye,    -   (b) one or more dextrins, and,    -   (c) an acid with a pKa of 4 or less.

In the reagent, it is preferred that the dye is present at aconcentration in the range of from about 0.001% to about 0.1% (w/v),preferably from about 0.005% to 0.05% (w/v). In use the reagent may bediluted, typically the ratio of reagent to diluent, e.g.protein-containing solution, will be in the range of from about 1:1 toabout 1:60. For detection of protein in solutions containing 25 μg/ml orless protein, a 1:1 volume ratio of reagent to protein-containingsolution could be used. For solutions with a higher proteinconcentration, e.g. 0.1 mg/ml to 2 mg/ml, a 1:60 volume ratio of reagentto protein-containing solution would be appropriate.

Useful acids have a pKa in the range of 0 to 4, preferably 3 or less sothat the reagent has a pH of −1 to 1; more preferably the acid will havea pKa in the range of from about 1 to about 3, so that the reagent has apH of 0 to 1. Many useful acids are identified in the Bradford patent(U.S. Pat. No. 4,023,933) and Gindler patent (U.S. Pat. No. 4,239,495),suitable acids include a phosphoric acid, a phosphorous (phosphonic)acid, periodic acid, selenic acid, maleic acid, oxalic acid anddichloroacetic acid. Phosphoric and phosphonic acids are preferred. Apreferred phosphonic acid is Nitrilotris (methylene) triphosphonic acid(NTP), a commercially available polybasic acid.

In a reagent according to the invention, when acid is present, it willgenerally be present at a concentration of from about 4% to about 20%preferably from about 4% to about 12%, preferably from about 7.5% toabout 9.5% (w/v). The reagent may be diluted in use such that the finalconcentration of acid will be in the range of from about 2% to about20%.

The acid can be a mixture of polybasic and monobasic acid, in suchmixtures it is preferred that the ratio of polybasic to monobasic acidis in the range of from about 2:1 to about 3:1. In the reagent, apolybasic/monobasic acid mixture is generally present at a concentrationin the range of from about 1 to about 15% (v/v), preferably of fromabout 2% to about 5% (v/v). For use, the reagent may be diluted to givea final concentration of the polybasic/monobasic acid mixture in therange of from about 0.5 to about 15%.

A reagent according to the invention comprises one or more dextrins,preferably selected from a linear dextrin (D), a cyclodextrin (CD), acycloamylose (CA) and derivatives thereof. A preferred reagent comprisesone or more cyclodextrins. Suitable linear dextrins comprise 6 or moreglucose units, preferably 10 or more glucose units, e.g. 15 glucoseunits; cyclodextrins will generally have 6 (α-CD), 7 (β-CD), or 8 (γ-CD)glucose units; cycloamyloses will generally comprise 8 or more glucoseunits. Suitable derivatives include heptakis 2,6-di-o-butylβ-cyclodextrin, carboxymethyl β-cyclodextrin and carboxymethylα-cyclodextrin.

Mixtures of dextrins may be used, for example mixtures of cyclicdextrins, such as two or more cyclic dextrins selected from α-CD, β-CDand γ-CD; two or more cyclic dextrins selected from α-CD, β-CD, γ-CD andCA; mixtures of linear and cyclic dextrins such a linear dextrin and oneor more of α-CD, β-CD and γ-CD; or a linear dextrin and one or more ofα-CD, β-CD, γ-CD and CA. Unless the context directs otherwise, the term“dextrin” as used herein encompasses dextrins and dextrin derivatives.

Some derivatives of cyclodextrin, dextrin and certain cycloamyloses mayact as surfactants and may be less suited for use in reagents, methodsand kits of the invention. Some dextrins at certain concentrations willinterfere with the certain dyes due to the surface-active properties ofthe dextrin. The dextrins and their respective interferingconcentrations for different dyes can be easily determined by thoseskilled in the art.

For a given protein, the choice of dextrin or mixture of dextrins usedmay be optimised and where one or more dextrin is used, the ratio may beadjusted to achieve the most effective conditions for detection and/orquantification of a given protein or protein sample.

In reagents of the invention the dextrin(s) will generally be present ata concentration in the range of from 0.01 to 200 mg/ml, preferably inthe range of from 0.5 to 50 mg/ml. Where mixtures of dextrins are usedthese concentrations relate to the total dextrin concentration. Dilutionof the reagent may be adjusted such that the final concentration ofdextrin is optimised for a given protein and protein concentration. Whena detergent is present in the protein sample, the choice andconcentration of dextrin(s) may be optimised for a given detergent and aparticular concentration of the detergent. Appropriate final dextrinconcentrations can be easily determined by those skilled in the art forexample by measuring absorbance or emission spectra, as appropriate, ofthe protein-dye complex in the presence of various concentrations of thedextrin or mixture of dextrins. For Coomassie brilliant blue G-250,absorption can be measured at the absorption peak, 595 nm.

A reagent according to the invention may further comprise a solubilisingagent, such as an alcohol, to maintain solubility of the dye-proteincomplex. The solubilising agent can be any agent that reduces or delaysprecipitation of the dye-protein complex.

One or more alcohols may be included in the reagent, suitable alcoholsinclude ethanol, methanol and propanol. Other appropriate alcohols arethose with good water solubility that show little or no behaviour asdetergents. When alcohol is present in the reagent, the concentration isgenerally from 0.1% to about 10% (v/v), preferably from about 0.1% toabout 5% (v/v), more preferably from about 1% to about 5% (v/v).

A reagent of the invention may comprise a detergent.

The reagent can be provided in a multipart system, e.g. as one or moreaqueous components, which are combined to form a reagent of theinvention. If provided in two parts, one part may comprise the dye,optionally acid and/or optionally alcohol, whilst the other may comprisethe dextrin(s). Each individual component has extended stability (forabout one year when kept refrigerated) and when mixed to form thereagent, the reagent itself is stable for more than 6 months when keptrefrigerated at 4° C. A reagent according to the invention may begenerated by combining one or more dextrin(s) with commerciallyavailable protein staining reagents, e.g. Bradford assay reagents orother Coomassie protein staining reagents. Commercially availableprotein staining reagents, methods and kits; in particular commerciallyavailable Bradford assay reagents methods and kits, can be adapted byinclusion of one or more dextrins in accordance with the invention.Examples of such commercially available kits include the following:

Pierce:

23236 Coomassie Plus—The Better Bradford Assay Kit (includes standards)

23238 Coomassie Plus—The Better Bradford Assay Reagent

23200 Coomassie (Bradford) Protein Assay Kit

23296 Coomassie (Bradford) Dry Protein Assay Plates 2×96 well

23596 Coomassie (Bradford) Dry Protein Assay Plates 5×96 well

BioRad:

500-0201EDU Quick Start Bradford Protein Assay Kit 1

500-0202EDU Quick Start Bradford Protein Assay Kit 2

500-0203EDU Quick Start Bradford Protein Assay Kit 3

500-0204EDU Quick Start Bradford Protein Assay Kit 4

500-0006EDU Bio-Rad Protein Assay Dye Reagent Concentrate

Sigma Aldrich:

B6916 Bradford Reagent (Sigma)

27813 Coomassie® protein assay reagent BioChemika (Fluka)

Conventionally, detection and quantitation of protein using someprotein-complexing dyes is subject to very significant interference fromdetergents; particularly adversely affected are the protein complexingdyes Coomassie blue G-250, Coomassie Red G-250, Coomassie Orange, Biuretreagent and Biuret reagent with Folin-Ciocalteu reagent. This inventionovercomes these difficulties. Without wishing to be bound by theory, itis believed that the detergent forms a complex with the dextrin and theaffinity of the detergent for the dextrin is higher than the affinity ofthe detergent for the dye. By using a suitable amount of a dextrin or amixture of dextrins, the detergent can be trapped in a dextrin-detergentcomplex, thereby, limiting the degree to which the detergent inhibitsthe protein-dye reaction.

Compared to currently available reagents for protein detection, reagentsof the invention can be used successfully when detergents are present inthe protein-containing samples. This is of great significance asreagents of the invention allow protein detection in an environment richin detergents or surfactants, such as may be required to solubilizemembrane proteins or to extract proteins directly from micro-organismsusing detergent rich solutions, e.g. commercially available extractionsolutions such as B-PER®, and CelLytic™.

The invention further provides a method of detecting protein comprisingcontacting a protein-containing sample with a solution comprising:

-   -   (a) a protein-complexing dye, and,    -   (b) one or more dextrins,        and detecting formation of a dye/protein complex.

For dyes that require strongly acidic conditions, the invention providesa method of detecting protein comprising contacting a protein-containingsample with a solution comprising:

-   -   (a) a protein-complexing dye,    -   (b) one or more dextrins, and,    -   (c) an acid with a pKa of 4 or less;        and detecting formation of a dye/protein complex.

Detecting the formation of dye/protein complex may comprise quantifyingthe amount of dye/protein complex formed, so as to determine theconcentration of protein in the sample.

In a further embodiment the invention provides a method of quantifyingprotein comprising contacting a sample containing protein with asolution comprising:

-   -   (a) a protein-complexing dye,    -   (b) one or more dextrin(s),        and quantifying dye/protein complex formation.

For dyes that require a strongly acidic environment, the inventionprovides a method of quantifying protein comprising contacting a samplecontaining protein with a solution comprising:

-   -   (a) a protein-complexing dye,    -   (b) one or more dextrin(s), and,    -   (c) an acid with a pKa of 4 or less;        and quantifying dye/protein complex formation.

The protein-containing sample can be a solution, or theprotein-containing sample can be provided on a support, such as a gel,sol, chromatography plate, filter paper, nitrocellulose membrane orresin.

Accordingly, in an additional embodiment the invention further providesa method of detecting protein comprising:

-   -   (a) providing a support comprising protein,    -   (b) contacting the protein with a solution comprising:        -   (i) a protein-complexing dye, and,        -   (ii) one or more dextrin(s),    -   and detecting dye/protein complex formation.

For protein complexing dyes that require acidic conditions, theinvention provides a method of detecting protein comprising:

-   -   (a) providing a support comprising protein,    -   (b) contacting the protein with a solution comprising:        -   (i) a protein-complexing dye,        -   (ii) one or more dextrin(s), and,        -   (iii) an acid with a pKa of 4 or less;    -   and detecting dye/protein complex formation.

Suitable protein complexing dyes, dextrins and, if required, acids forinclusion in the solution used in methods of the invention are describedabove. The protein-complexing dye, one or more dextrin(s) and, ifpresent, acid with a pKa of 4 or less, can be provided by a reagentaccording to the invention, which may be diluted to form the solution.The solution may comprise a solubilising agent such as an alcohol asdescribed herein.

The support can be a gel, sol, chromatography plate, filter paper,nitrocellulose membrane or resin. The support may comprise a detergent.Using methods of the invention contacting can be performed in thepresence of a detergent. These methods are particularly suitable fordetecting protein in polyacrylamide gel, agarose gel or polymercomposite gel, for example when a protein sample has been separatedusing an electric field, e.g. by electrophoresis.

The reagents and methods described herein for the detection andquantitative determination of protein in gels, such as those producedfollowing separation using an electric field, e.g. by electrophoresis,simplify conventional procedures so that washing procedures to removedetergents such as SDS and excessive stain (background stain) are nolonger required.

Typically, the methods are carried out at room temperature.

In methods of the invention detecting formation of a dye protein complexmay comprise detecting a change in absorption or emission spectra of thedye/protein complex. In some instances, a colour change may be detected;for example when using Coomassie brilliant blue dyes such as G-250.Colour changes may be detected using conventional apparatus, such as acolorimeter, for example capable of measuring absorbance at a wavelengthin the range of from 570 nm to 620 nm.

For dyes that undergo a change in absorption spectra on formation of aprotein/dye complex, detecting can be performed by measuring absorbance,for example using a spectrophotometric method. Conventional apparatusmay be used for spectrophotometric analyses, such as a UV/VISSpectrophotometer with a wavelength range of from 400 to 700 nm.

For dyes that undergo a change in emission spectra on formation of aprotein/dye complex, detecting can be performed by measuring emission,for example using a spectrofluorimeter (luminescent spectrometer),suitably with a wavelength range of 190 nm to 800 nm.

Detecting the protein/dye complex may comprise quantifying the amount ofprotein/dye complex present so as to determine the amount orconcentration of protein. Quantifying can be performed by methods thatcomprise measuring a change in absorption or emission spectra of thedye/protein complex. Quantifying may comprise for example measuring acolour change. As described absorbance can be measured by aspectrophotometric method and change in absorbance over time may bemeasured. Absorbance is generally measured at a wavelength in the rangeof from about 400 to about 700 nm.

For Coomassie brilliant blue G-250, absorbance is measured at awavelength of about 595 nm, the absorbance maximum for this dye whencomplexed to protein. When using Coomassie brilliant blue G-250, proteincan be detected by monitoring of the increase in absorbance at 595 nmdue to formation of the dye/protein complex.

To determine protein concentration, the absorbance or emission measuredcan be compared with a standard value, standard set of values, orstandard curve. The results are highly reproducible and accurate asshown in the Examples.

Because of the high sensitivity displayed using reagents and methods ofthe invention, protein concentrations can be selected which are as lowas approximately 0.1 μg per 1 ml of sample. Moreover, the time requiredfor such accurate and sensitive determinations is less than about 2minutes per sample in contrast to 30-40 minutes generally required fortraditional Lowry or Biuret type assays. Consequently, methods of thisinvention are highly amenable to automation and analysis of largenumbers of samples.

The invention further provides kits for detecting and/or quantifyingprotein, the kits comprising one or more dextrin(s). Kits for detectingand/or quantifying protein may comprise one or more dextrins and aprotein-complexing dye. Additionally, a kit may comprise one or moreacid(s) and/or alcohol(s) as described herein. A kit for detectingand/or quantifying proteins in accordance with the invention maycomprise a reagent of the invention, which may be provided as amultipart system wherein the components are mixed to form a reagent ofthe invention.

The invention further provides the use of one or more dextrin(s) toenhance formation of a protein-binding dye/protein complex in thepresence of a detergent.

Furthermore, the invention provides the use of one or more dextrins toreduce interference of a detergent in formation of a protein-bindingdye/protein complex in the presence of a detergent.

The invention yet further provides the use of one or more dextrins toalter the optical properties of a dye, such as a protein-complexing dye,in the presence of a detergent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Standard curves for protein samples without detergent

Both methods give reasonable linear response for samples free ofdetergent. The slope of the curve and the correlation coefficient arecomparable for both methods indicating the dextrins included in thereagent do not interfere with protein-dye binding.

FIG. 2: Standard curves for protein samples including detergent (0.25%CTAB). Only the reagent including dextrins gives a linear response withthe slopes and the correlation coefficient being comparable to theslopes and correlation coefficient for samples without detergent.

FIG. 3: Detection of protein in polyacrylamide gel

The following samples were run on each gel:

Lane 1 Molecular weight standard, Mark 12™

Lane 2 Beta-lactoglobulin 0.08 mg/ml

Lane 3 Beta-lactoglobulin 0.16 mg/ml

Lane 4 Beta-lactoglobulin 0.31 mg/ml

Lane 5 Beta-lactoglobulin 0.63 mg/ml

Lane 6 Beta-lactoglobulin 1.25 mg/ml

Lane 7 Beta-lactoglobulin 2.5 mg/ml

Lane 8 Empty

Lane 9 Beta-lactoglobulin 5 mg/ml

Lane 10 Empty

Lane 11 Beta-lactoglobulin 10 mg/ml

Lane 12 Molecular weight standard

Gel B was stained with solution B, gel A was stained with solution A, asdescribed in Example 3. The gels were photographed after 1 hour 45minutes incubation in the staining solutions.

EXAMPLES Example 1 Preparation of Bradford Reagent

To 100 mg Coomassie Brilliant Blue (G-250) was added 47 g ethanol, 85 gphosphoric acid and 850 g water. This solution was mixed for 20 minutes,to ensure all components were dissolved, resulting in a reagentcomprising 0.01% (w/v) Coomassie Brilliant Blue G-250, 4.7% ethanol(w/v) and 8.5% (w/v) phosphoric acid. To this solution differentdextrins were added as indicated in the specific examples.

Bradford Assay (Standard Method)

Five microliters of sample solutions containing from 0.1 mg/ml to 1.5mg/ml protein and/or from 0.00% to 0.5% detergent were pipetted into thewells of 96-well microtitre plates. To this was added 300 microliters ofBradford reagent. The absorbance was measured at 595 nm.

Reducing Detergent Interference

Five different detergents were used in the experiment, namely sodiumdodecylsulphate (SDS) (anionic), Cetyltrimethylammonium bromide (CTAB)(cationic), TWEEN™-20 (non-ionic), TRITON™-X 100 (non-ionic) and Brij-35(non-ionic). Four different dextrins were used in the experiment, namelydextrin-15 (D15), alpha-cyclodextrin (α-CD), beta-cyclodextrin (β-CD)and gamma-cyclodextrin (γ-CD), an equimass mixture of these dextrins(MIX) was also used. In addition, different cycloamylose concentrationswere also tested.

A) Bradford assay containing a total of 100 mg/ml dextrin(s) orsaturated concentration of the various dextrin(s).

Table of absorbance measured at 595 nm blanked against a water sample.No dextrin D15 α-CD β-CD γ-CD MIX SDS (0.5% w/v) 0.326 0.007 0.013 0.0890.038 0.047 CTAB (0.25% w/v) 0.785 0.024 0.003 0.067 0.015 0.020TWEEN-20 (0.25% 1.027 0.453 0.134 0.077 0.344 0.041 w/v) TRITON-X 1000.677 0.101 0.034 0.017 0.022 0.003 (0.10% w/v) Brij-35 (0.50% w/v)0.231 0.035 0.015 0.019 0.143 0.018

B) Bradford assay containing a total of 10 mg/ml dextrin(s) of thevarious dextrin(s).

Table of absorbance measured at 595 nm blanked against a water sample.No dextrin D15 α-CD β-CD γ-CD MIX SDS (0.5% w/v) 0.326 0.051 0.003 0.0250.005 0.000 CTAB (0.25% w/v) 0.785 0.184 0.026 0.029 0.164 0.008TWEEN-20 (0.25% 1.027 0.855 0.167 0.171 0.625 0.376 w/v) TRITON-X 1000.677 0.076 0.003 0.006 0.019 0.095 (0.10% w/v) Brij-35 (0.50% w/v)0.231 0.046 −0.02 0.001 0.007 0.026

C) Bradford assay containing a total of 1 mg/ml dextrin(s) of thevarious dextrin(s).

Table of absorbance measured at 595 nm blanked against a water sample.No dextrin D15 α-CD β-CD γ-CD MIX SDS (0.5% w/v) 0.326 0.195 0.010 0.0560.079 0.020 CTAB (0.25% w/v) 0.785 0.674 0.163 0.176 0.603 0.407TWEEN-20 (0.25% 1.027 0.999 0.842 0.803 0.952 0.947 w/v) TRITON-X1000.677 0.248 0.288 0.040 0.032 0.003 (0.10% w/v) Brij-35 (0.50% w/v)0.231 0.098 0.003 0.033 0.040 0.002

D) Bradford assay containing various concentrations of cycloamylose(CA).

Table of absorbance measured at 595 nm blanked against a water sample.CA CA No dextrin 10 mg/ml 1 mg/ml SDS (0.1% w/v) −0.063 −0.072 −0.023CTAB (0.1% w/v) 0.412 −0.081 0.003 TWEEN-20 (0.1% 0.462 0.074 0.352 w/v)TRITON-X 100 0.677 −0.024 0.410 (0.1% w/v) Brij-35 (0.1% w/v) −0.024−0.025 −0.011

The absorbance at 595 nm provides an indication of the amount of theblue form of the Coomassie G-250 dye. At a given concentration of aparticular detergent, a high absorbance value indicates high background,due to the presence of the detergent, such that that the blue colourdetected is not representative of the amount of protein-dye complexpresent and thus is not representative of the protein concentration. Itcan be seen that for each of the detergents tested, the presence of adextrin or dextrins in the solution resulted in a lower absorbancevalue, indicating that there is less background interference and thatthe presence of dextrin increases the sensitivity and accuracy ofprotein detection. For a given detergent the optimal choice of dextrinand concentration of dextrin can be determined by measuring absorbanceat the absorbance peak of the dye/protein complex in the presence ofvarious concentrations of dextrins and combinations thereof.

Example 2 Linearity of Standard Curves in Presence of Detergents

A dilution series of two different proteins, bovine serum albumin (BSA)(Pierce, 23209) and bovine immunoglobulin (IgG) (Sigma, I5506) wascreated in the concentration range 0.1 mg/ml to 1.5 mg/ml. CTAB waschosen as detergent and was added to the protein samples to aconcentration of 0.25% (w/v). Bradford assays were performed withBradford reagent containing 0.25 mg/ml dextrin-15 (D15), 0.25 mg/mlalpha-cyclodextrin (α-CD), 0.25 mg/ml beta-cyclodextrin (β-CD) and 0.25mg/ml gamma-cyclodextrin (γ-CD), these were compared with Bradford assayperformed with Bradford reagent containing no dextrins.

Example 3 Detection of Protein in Polyacrylamide Gel

Preparation of Gel Staining Solutions

To 80 mg Coomassie Brilliant Blue (G-250) was added 50 g ethanol, 80 gphosphoric acid and 850 g water. The solution was mixed for 20 minutesto ensure all components were dissolved—this was solution A. Followingthis, solution B was made by adding 250 mg dextrin-15, 250 mgalpha-cyclodextrin (α-CD), 250 mg beta-cyclodextrin (β-CD) and 250 mggamma-cyclodextrin (γ-CD) to 100 ml of solution A.

Preparation of the Samples and Gels

A dilution series of beta-lactoglobulin (BLG) (Sigma, L-0130) wasprepared in the concentration range 0.08 mg/ml to 10 mg/ml. Thirtymicroliters of the sample was diluted with ten microliters Nupage LDSSample buffer (Invitrogen, NP0007). Duplicate gels were prepared,fifteen microliters of each sample was loaded onto each gel (Nupage 10%Bis-Tris, Invitrogen, NP0302). Ten microliters of the Mark 12™ molecularweight standard (Invitrogen, LC5677) was also loaded in the gel. Thegels were run at constant voltage (200V) for 35 min in standard MESbuffer. Following this, one gel was submerged in 25 ml of solution B (astaining solution which consisted of 25 ml of solution A as describedabove containing a mixture of dextrins as follows: 2.5 mg/ml dextrin-15(D15), 2.5 mg/ml alpha-cyclodextrin (α-CD), 2.5 mg/ml beta-cyclodextrin(β-CD) and 2.5 mg/ml gamma-cyclodextrin (γ-CD)). The other gel wassubmerged in a staining solution consisting of 25 ml of solution A.After 1 hour 45 minutes incubation in the staining solutions, the gelswere photographed (FIG. 3).

The invention claimed is:
 1. A method of detecting and/or quantifyingprotein comprising contacting a protein-containing sample with asolution comprising: (a) a protein complexing dye; and (b) one dextrinselected from α-cyclodextrins, γ-cyclodextrins, linear dextrins andderivatives thereof, or two or more dextrins; and detecting and/orquantifying dye/protein complex formation, wherein the protein is in thepresence of a detergent.
 2. The method according to claim 1, wherein theprotein is in solution or the protein is provided on a support.
 3. Themethod according to claim 2, wherein the support is a gel, sol,chromatography plate, filter paper, nitrocellulose membrane or resin. 4.The method according to claim 3, wherein the support is a polyacrylamidegel or agarose gel and the protein has been subjected to separationusing an electric field.
 5. The method according to claim 2, wherein thesupport comprises a detergent and/or wherein contacting is performed inthe presence of a detergent.
 6. The method according to claim 1, whereindetecting and/or quantifying comprises detecting a change in absorptionor emission spectra of the dye/protein complex.
 7. The method accordingto claim 6, wherein detecting and/or quantifying comprises detecting acolour change.
 8. The method according to claim 6, wherein theprotein-complexing dye is a Coomassie G-250 dye and absorbance ismeasured at a wavelength of about 595 nm.
 9. The method according toclaim 1, wherein quantifying is performed by measuring absorbance overtime.
 10. The method according to claim 6, wherein the absorbance oremission measured is compared with a standard value, standard set ofvalues, or standard curve.
 11. The method according to claim 1, furthercomprising providing a support comprising protein, prior to thecontacting step.
 12. A method comprising contacting a protein-containingsample in the presence of a detergent with a protein binding dye, andone dextrin selected from α-cyclodextrins, γ-cyclodextrins, lineardextrins and derivatives thereof; and detecting and/or quantifyingdye/protein complex formation, wherein the protein is in the presence ofa detergent.
 13. A method of detecting and/or quantifying proteincomprising contacting a protein-containing sample in the presence of adetergent with a protein binding dye, and two or more dextrins; anddetecting and/or quantifying dye/protein complex.
 14. The methodaccording to claim 1, wherein the one dextrin or two or more dextrinsare provided as a reagent comprising a protein-complexing dye and onedextrin.
 15. The method according to claim 14, wherein the reagent is areagent concentrate.
 16. The method according to claim 14, wherein thereagent further comprises an acid with a pKa of 4 or less.
 17. Themethod according to claim 14, wherein the reagent further comprises asolubilizing agent.
 18. The method according to claim 14, wherein thereagent does not comprise a protein.
 19. The method according to claim14, wherein the dye is a Coomassie dye.
 20. The method according toclaim 1, wherein the one dextrin, or two or more dextrins concentrationis in the range of from about 0.01 to about 200 mg/ml.
 21. The methodaccording to claim 14, wherein the reagent further comprises one or morealcohols, and wherein the alcohol concentration is from about 0.1% toabout 10% v/v.
 22. The method according to claim 13, wherein the mixtureof two or more dextrins is selected from the group consisting ofα-cyclodextrins, β-cyclodextrins, γ-cyclodextrins, cycloamylose, lineardextrins and derivatives thereof.
 23. The method according to claim 14,wherein the reagent is provided as a multipart system.
 24. A method ofdetecting and/or quantifying protein comprising contacting aprotein-containing sample with a solution comprising: (a) aprotein-complexing dye, wherein the dye is a Coomassie dye; and two ormore dextrins; and detecting and/or quantifying dye/protein complexformation, wherein the protein is on a support.
 25. A method accordingto claim 24, wherein the dye is a Coomassie dye.
 26. A method accordingto claim 24, wherein the support is a gel, sol, chromatography plate,filter paper, nitrocellulose membrane or resin.
 27. The method accordingto claim 26, wherein the support is a polyacrylamide gel or agarose geland the protein has been subjected to separation using an electricfield.
 28. The method according to claim 24, wherein the supportcomprises a detergent and/or wherein contacting is performed in thepresence of a detergent.
 29. A method of detecting and/or quantifyingprotein comprising contacting a protein-containing sample with asolution comprising: (a) a protein-complexing dye; and (b) one dextrinselected from α-cyclodextrins, γ-cyclodextrins, linear dextrins andderivatives thereof; and detecting and/or quantifying dye/proteincomplex formation, wherein the protein is on a support.
 30. A methodaccording to claim 29, wherein the dye is a Coomassie dye.
 31. Themethod according to claim 30, wherein the Coomassie dye is a CoomassieG-250 dye.
 32. A method according to claim 29, wherein the support is agel, sol, chromatography plate, filter paper, nitrocellulose membrane orresin.
 33. The method according to claim 32, wherein the support is apolyacrylamide gel or agarose gel and the protein has been subjected toseparation using an electric field.
 34. The method according to claim29, wherein the support comprises a detergent and/or wherein contactingis performed in the presence of a detergent.